Nitric oxide and its biomedical significance

ABSTRACT

The invention relates to nitric oxide (NO) stimulating extracts from various plants. Such extracts contain compounds known as healthin I and healthin II. Specifically, the invention provides partially purified plant extracts that have NO stimulating activity, methods of isolating and partially purifying such extracts from plant materials. In addition, the invention provides methods and materials for treating diseases and conditions that require modification of cellular levels of NO, for example, diseases and conditions involving inflammation.

REFERENCE TO PRIOR APPLICATION

This application claims the benefit of co-pending P.C.T. Application No.PCT/US2003/25966, filed on Aug. 19, 2003, and U.S. ProvisionalApplication Ser. No. 60/405,787, filed on Aug. 23, 2002, and herebyincorporated herein by reference.

INVENTION FIELD

This invention discloses materials and methods involved in theproduction of nitric oxide. Specifically, the invention relates to lowmolecular weight, water soluble, molecules isolated from plant tissueand materials and the use of these molecules to induce nitric oxideproduction in mammalian cells and tissues.

INVENTION BACKGROUND

Nitric oxide (NO) is a major signaling molecule in the mammalian immune,cardiovascular and nervous systems.^(18,26,37,56,57,109) NO produced atone site can have an effect on tissues at a distance.^(24,70) NO isproduced from L-arginine by the enzyme, nitric oxide synthase(NOS).^(55,57) NOS occurs in three forms: endothelial (e), neuronal (n),and inducible (i) NOS. The first two forms are constitutively expressedand Ca²⁺ dependent. Inducible (i) NOS is Ca²⁺ independent. The threeforms of NOS are encoded for on three distinct genes on chromosomes, 7,12, and 17, respectively.^(18,26,37,54) In general, n- and e-NOS dependon intracellular calcium transients and release NO in the nM range,whereas iNOS, following an induction/latency period, can release NO inthe μM range for extended periods oftime.^(18,26,28,37,56,57,70,105,109) The presence of constitutive andinducible forms of NOS suggest that they may have distinct functions.

c- and i- NOS can be distinguished on the basis of the length of timenecessary to see an increase in levels of NO and the length of timethese elevated levels can be maintained. NO derived from cNOS may occurin two functional forms: the first is always present at low “tonal” or“basal” levels; this basal level can be slightly increased for a shorttime in response to certain signals, e.g., acetylcholine (ACH).⁵⁶ Thisbrief enhanced release of cNOS derived NO can have profoundphysiological actions, which are evident long after NO has returned toits basal level, for a longer period of time.⁵⁰ For example, endothelialcells briefly exposed to morphine and eNOS change their shape fromelongated to round, a process that takes several hours.⁵⁰

iNOS is induced by various signal molecules, e.g., proinflammatorycytokines.^(57,73,105) The induction of i-NOS is usually seen after a3-4 hour delay; iNOS is capable of producing NO for 24-48hours.^(73,105) These data suggest that NO is always present and thatthe levels of NO can be regulated either rapidly or slowly depending onthe organism's needs. The presence of different regulatory processesimplies that NO has different functions, and/or that the levels of NOmust be progressively increased in order for it to exert its function.

NO functions as a vascular, immune and neural signal molecule and alsohas general antibacterial, antiviral actions and the ability todown-regulated proinflammatory events.^(38-39,41-42,60,90,105-106) Inthe vascular and immune system, one of the key stages in the immuneresponse is the recruitment and activation of leukocytes by theendothelium. Leukocyte activation by the endothelium occurs in stages.The initial step is the attraction of the leukocytes to the endothelium.This is followed by increased leukocyte adhesion and change in shape andfinally migration across the endothelium.⁹⁰ These cellular changes areaccompanied by scheduled changes in synthesis of molecules that regulatecell-matrix interactions.^(3,46,52,87)

Normally, non-activated leukocytes roll along the endothelium. Theinteraction between the two cell types is loose and reversible andmediated by a family of adhesion molecules known as selectins.Activation of leukocytes occurs in response to the release of severalchemoattractants including leukotriene B₄ and interleukin 8 (IL-8). Inthe presence of these agents, immunocytes cease to roll, becoming“activated”: they start to flatten and adhere with greater strength tothe endothelial lining. Activation is mediated by a family of adhesionmolecules call the integrins, such as ICAM-1 and VCAM-1. Adherentimmunocytes are able to undergo transendothelial migration in thepresence of PECAM-1.^(3,46,52,87) This immunocyte-endothelialinteraction is down-regulated by NO. NO inhibits platelet and neutrophilaggregation and can diminish the adherence and level of activation ofleukocytes and endothelial cells.^(41,1,50, 109) NOS inhibitors increaseplatelet adhesion and enhance leukocyte adhesion.^(72,82) NO plays asimilar role involving the microglia cells of the nervous system'simmune response.^(83,84)

The central nervous system (CNS) is unique in that it uses all threeisoforms of NOS to produce NO. The constitutive isoforms e- and n- NOSare found in the normal CNS; however, iNOS is not expressed in thehealthy CNS.²⁰ Pathological states, e.g., trama, cerebral ischemia andneuronal diseases, increase the levels of e- and nNOS and induce iNOSactivity.²¹ cNOS derived NO has the ability to down-regulateproinflammatory events via inhibition of NF-κB activation ofproinflammatory cytokines.

NO upregulates several enzymes involved in immunoregulation, includingneutral endopeptidese 24.11 (CALLA, acute lymphoblastic leukemicantigen, enkephalinase) or CD10.⁷⁶ Thus, cNOS derived No stimulatesenzymes that process protein gene products, implying a link betweensignaling processes involving NO and naturally occurring antibacterialpeptides. No controls and regulates enzymes that are responsible forliberating these crucial molecules that have a proactive protectivefunction.¹⁰¹

Evidence has also been provided that NO plays a role in neurotransmitterrelease.¹⁰² Morphine and cNOS derived NO release growth hormone and ACTHfrom rat brain fragments; these neuropeptides are involved in the stressresponse. Thus, NO is involved in vasodilation, antibacterial andantiviral responses, signal molecule release and inhibition ofimmunocyte adherence to the endothelium.

There appears to be a tonal or basal level of NO that is physiologicallysignificant. Endothelia from non-insulin dependent diabetics do notexhibit a tonal level of NO¹¹⁷ and in these individuals vascular diseasecauses disability and eventual death.¹⁴ A number of researchers haveattributed vascular disease in part to alterations associated witheNOS-derived NO and some have speculated this may be due to enhancedfree radical generation.⁵⁹ Decreases in basal NO levels may alsocontribute to enhanced platelet function and variousneuropathies.^(32,68)

Thus, it appears that tonal or basal NO levels are important in limitingthe degree of excitation of nervous, immune and vascular tissues. Thistonal NO may manifest itself via effects on adhesion-mediated processesvia NF-κB. Estrogen may exert it beneficial vascular protective actionsvia these processes as well, since it also releases cNOS derivedNO.^(70,99) Strengthening this hypothesis in the finding of thecannabinoid CB1 receptor type on mammalian endothelial cells^(118,119)and the finding of a mu opiate receptor on human vascular endothelialcells. (Three general classes of cell surface opioid receptors (kappa,delta and mu) have been described. Receptors exhibiting high bindingspecificity for morphine have been designated mu opioid receptors.)Detailed analysis has revealed the existence of multiple mu opioidreceptor subtypes. Isolated nucleic acid sequences encoding various mureceptors and polypeptides comprising mu receptors (and referred to hereas “mu3 opioid receptor(s)”) are disclosed in detail in PCT PatentPublication WO 99/24471, published 20 May 1999. See also, MolecularIdentification and Functional Expression of μ₃, a Novel AlternativelySpliced Variant of the Human μ Opiate Receptor Gene.

Consequently, promoting NO generation at normal or slightly enhancedlevels may have significant health value. While the health promotingeffects of many plants are well known, how and why this occurs at amolecular level is less understood. See Stefano and Miller,Communication between animal cells and the plant foods they ingest:Phyto-zooidal dependencies and signalling (Review), Intl J Mol Medicine10: 413-21 (2002) incorporated by reference herein.

INVENTION SUMMARY

The invention relates to nitric oxide (NO) stimulating extracts fromvarious plants. Such extracts contain compounds known as healthin I andhealthin II. Specifically, the invention provides partially purifiedplant extracts that have NO stimulating activity, methods of isolatingand partially purifying such extracts from plant materials. In addition,the invention provides methods and materials for treating diseases andconditions that require modification of cellular levels of NO, forexample, diseases and conditions involving inflammation.

The invention is based on the discovery of a class of agents identifiedby extraction and chemical analysis of certain plant species that arecapable of stimulating NO production in mammalian cells and tissues.These NO stimulating agents stimulate the production of constitutivenitric oxide synthase in mammalian vascular endothelial cells and/orneuronal cells in culture.

Accordingly, the invention provides active, chemical agents isolatedfrom plant tissue and materials that stimulate the production of nitricoxide in pedal ganglia and human endothelial cells. Partially purifiedextracts from any of the plants listed below contain various amounts ofthe active agents.

In addition, the invention provides methods and materials foridentifying additional NO stimulating botanical agents from other plantshaving such activity and methods and materials useful in the treatmentof diseases and conditions requiring modification of cellular levels ofNO.

These botanical agents of the invention are additionally characterizedas having:

-   -   (i) the ability to stimulate nitric oxide release in the range        of 15 nM to 100 nM in pedal ganglia cells;    -   (ii) the ability to stimulate nitric oxide release in the range        of 50 nM to 100 nM in endothelial cells;    -   (iii) a single major peak on high performance liquid        chromatographic analysis in 10 nM sodium chloride, 0,5 mM EDTA,        100 mM sodium acetate and 50% acetonitrile, pH 5.0.

The NO stimulating botanical agents of the invention may be furthercharacterized by being water soluble and having a molecular mass ofbetween about 50 and about 5000 Daltons, or between about 50 and about2500 Daltons, or between about 50 and about 1000 Daltons, or betweenabout 50 and about 500 Daltons.

The botanical agents of the invention can be extracted from plantsselected from the group consisting of Agropyrum spp., Salix alba, Alliumvineale, Petroselinium crispum, Taraxacum officinale, Sesamum indicum,Medicago spp., Piper methysticum, Anthemis spp., Tumera diffusa,Verbascum densiflorum, Ocimum spp., Maranta arundinaceae, CoriandrumSativum, Artemesia dracunculus, Lavendula augustifolia, Mentha pulegium,Centella asiatica, Ginko biloba, and Vitus vinifera.

Accordingly, one aspect of the invention is a pharmaceutical compositionconsisting of low molecular weight, water soluble, extract of at leastone of the following plants: Allium vineale, salix alba, Agropyrum spp.,Petroselinium crispum, Taraxacum officinale, Sesamum indicum, Medicagospp., Piper methysticum, Anthemis spp., Turnera diffusa, Verbascumdensiflorum, Ocimum spp., Maranta arundinaceae, Coriandrum sativum,Artemesia dracunculus, Lavendula augustifolia, Mentha pulegium, Centellaasiatica, Ginko biloba and Vitis vinifera, which extracts have nitricoxide stimulating ability in mammalian cells. These extracts areadditionally characterized as having the ability to stimulate nitricoxide release in the range of 15 nM to 100 nM in pedal ganglia cellsand/or the ability to stimulate nitric oxide release in the range of 50nM to 100 nM in endothelial cells. These extracts are also characterizedby having components greater than 5000 Daltons removed, i.e., bycomprising low molecular weight water soluble components in the range ofabout 50 to 5000 Daltons. More preferably, components greater than 2500Daltons are removed and water soluble components in the range of about50 to about 2500 Daltons are included. Most preferably, componentsgreater than 1000 Daltons are removed and water soluble components inthe range of about 50 to about 1000 Daltons are included. Especiallypreferred are extracts having water soluble components in the range ofabout 50 to about 500 Daltons. These extracts are additionallycharacterized as exhibiting a single major peak on high performanceliquid chromatographic analysis in 10 nM sodium chloride, 0,5 mM EDTA,100 mM sodium acetate and 50% acetonitrile, pH 5.0.

These extracts may be dried and formed into pharmaceutical compositionscomprising powders, tablets, poltices, pastes, creams, plasters,capsules and the like, with or without pharmceutically acceptableexcipients and/or adjuvants, in accordance with known methods andtechniques, for example, as detailed in Remington's PharmaceuticalSciences, A. R. Gennaro, ed., Mack Publ. Co. Easton, Pa., 1985.

Another aspect of the invention is to provide a method for identifyingand isolating low molecular weight extracts of at least one of theplants set forth above, which extracts exhibit NO stimulating activityin mammalian cells.

A further aspect of the invention is to provide a method of using lowmolecular weight extracts of at least one of the plants set forth above,which extracts exhibit NO stimulating activity in mammalian cells.

An additional aspect of the invention is a method for preparing an NOstimulating extract of at least one of the plants set forth above bypreparing an aqueous extract having components being water soluble andhaving a molecular mass of between about 50 and about 5000 Daltons, orbetween about 50 and about 2500 Daltons, or between about 50 and about1000 Daltons, or between about 50 and about 500 Daltons.

Other features and advantages will be apparent from the followingdetailed description, drawings and claims.

DRAWING DESCRIPTIONS

FIG. 1 is a reproduction of the HPLC chromatogram of the wheat grassextraction detailed in Example 1.

FIG. 2 is a reproduction of the HPLC chromatogram of the white willowbark extraction detailed in Example 2.

FIG. 3 is a reproduction of the data print out from the massspectrometric analysis detailed in Example 3.

FIG. 4 is a reproduction of the data print out from the massspectrometric analysis detailed in Example 4.

FIG. 5 and FIG. 6 illustrate the results of the pedal ganglia andendothelial cell stimulation by Agropyrum spp. plant extracts asdetailed in Example 5.

FIG. 7 and FIG. 8 illustrate the results of the pedal ganglia andendothelial cell stimulation by Salix alba extracts as detailed inExample 6.

FIG. 9 illustrates the results of the pedal ganglia cell stimulation byTaracum officinale extracts as detailed in Example 7.

FIG. 10 illustrates the results of the pedal ganglia cell stimulation byVitus extracts as detailed in Example 8.

DETAILED DESCRIPTION

The invention provides active, chemical agents isolated from planttissue and materials that stimulate the production of nitric oxide inpedal ganglia and human endothelial cells. Low molecular weight, watersoluble, extracts from any of the plants listed below contain variousamounts of the active chemical agents that stimulate production of NO.In addition, the invention provides methods and materials foridentifying and isolating additional NO stimulating botanical agentsfrom other plants having such activity and methods and materials usefulin the treatment of diseases and conditions requiring modification ofcellular levels of NO.

These botanical extracts of the invention are additionally characterizedas having:

-   -   (iv) the ability to stimulate nitric oxide release in the range        of 15 nM to 100 nM in pedal ganglia cells;    -   (v) the ability to stimulate nitric oxide release in the range        of 50 nM to 100 nM in endothelial cells; and/or    -   (vi) a single major peak on high performance liquid        chromatographic analysis in 10 nM sodium chloride, 0,5 mM EDTA,        100 mM sodium acetate and 50% acetonitrile, pH 5.0.

The NO stimulating botanical agents of the invention may be furthercharacterized by being water soluble and having a molecular mass ofbetween about 50 and about 5000 Daltons, or between about 50 and about2500 Daltons, or between about 50 and about 1000 Daltons, or betweenabout 50 and about 500 Daltons.

The extracts of the invention can be isolated from plants selected fromthe group consisting of Allium vineale, salix alba, Agropyrum spp.,Petroselinium crispum, Taraxacum officinale, Sesamum indicum, Medicagospp., Piper methysticum, Anthemis spp., Tumera diffusa, Verbascumdensiflorum, Ocimum spp., Maranta arundinaceae, Coriandrum sativum,Artemesia dracunculus, Lavendula augustifolia, Mentha pulegium, Centellaasiatica, Ginko biloba and Vitis vinifera.

The method of isolating and extracting to obtain the active componentcomprises homogenizing dried plant material in an acidic solutionfollowed by alcohol extraction and centrifugation for filtration toseparate the solid material. The supernatant is dried and then dissolvedin an aqueous solution containing trifluroacetic acid and subjected tosolid phase extraction. The elute is collected and further purifiedusing high performance liquid chromatography. The extracted activecomponent can be further identified and characterized by massspectrometric analysis.

The method of identifying NO stimulating botanical agents of theinvention comprises homogenizing dried plant material in an acidicsolution followed by alcohol extraction and centrifugation, again forfiltration to separate the solid material. The supernatant is dried andthen dissolved in an aqueous solution containing trifluroacetic acid andsubjected to solid phase extraction. The elute is collected and furtherpurified using high performance liquid chromatography and the extracted,low molecular weight, NO stimulating agents are identified by massspectrometric analysis.

These extract are useful in the preparation of pharmaceuticalcompositions for treating antimicrobial infections such as bacterialinfections and viral infections, and asthma, and/or other inflammatoryconditions in mammals, especially in humans. The extracts, as detailedbelow, exhibit antibacterial, antinflammatory and anticancer effects.Consequently, pharmaceutical compositions comprising such extracts canbe administered in the treatment various diseases and conditions inwhich antibacterial, antinflammatory or anticancer effects are desired,such as for example, in microbial infections. Alternatively, thepharmaceutical compositions of the invention may be employed asprophylactics. To form the extracts into pharmaceutical compositions,they may be dried, alone or in various combinations, and formed intopharmaceutical compositions comprising powders, tablets, poltices,pastes, creams, plasters, capsules and the like, with or withoutpharmnceutically acceptable excipients and/or adjuvants, in accordancewith well known methods and techniques, for example, as detailed inRemington's Pharmaceutical Sciences, A. R. Gennaro, ed., Mack Publ. Co.Easton, Pa., 1985.

The invention will be further described in the following examples,without limiting the scope of the invention as described in the claims.In the examples, the plant extracts were made from the leaves of theplant, unless otherwise specified.

EXAMPLES Example 1 Extraction of Healthin I from Wheat Grass

One grams of dried wheat grass plants, Agropyron spp. were homogenizedin 1N HCl (0.5 g/ml). The resulting homogenates were extracted with 5 mlchloroforrn/isopropanol 9:1. After 5 min at room temperature,homogenates were centrifuged at 3000 rpm for 15 min. The supernatant wascollected and dried with a Centrivap Console (Labconco, Kansas City,Mo.). The dried extract was then dissolved in 0.05% trifluoroacetic acid(TFA) water before solid phase extraction. Samples were loaded on aSep-pak Plus C-18 cartridge (Waters, Milford, Mass.) previouslyactivated with 100% acetonitrile and washed with 0.05% TFA-water.Morphine elution was performed with a 10% acetonitrile solution(water/acetonitrile/TFA, 89.5%: 10%: 0.05%, v/v/v). The eluted samplewas dried with a Centrivap Console and dissolved in water prior to highperformance liquid chromatography analysis (HPLC).

Reverse phase HPLC analysis using a gradient of acetonitrile wasperformed on a C-18 Unijet microbore column (BAS, West Lafayette, Ind.)using a Waters 626 pump (Waters, Milford, Mass.). 0.025 g dry weight ofthe wheat grass from the above-described extraction was used. The mobilephases were: Buffer A: 10 mM sodium chloride, 0.5 mM EDTA, 100 mM sodiumacetate, pH 5.0; Buffer B: 10 mM sodium chloride, 0.5 mM EDTA, 100 mMsodium acetate, 50% acetonitrile, pH 5.0. A flow splitter (BAS), withsplit ratio 1/9 was used to provide the low volumetric flow ratesrequired for the microbore column. Operating the pump at 0.5 ml/minyielded a microbore column flow rate of approximately 50 μl/min. Theinjection volume was 5 μl. The running conditions were: 0 min, 0% BufferB; 10 min, 5% Buffer B; 25 min, 50% Buffer B; 30 min, 100% Buffer B.Both buffers were filtered through a Waters 0.22 μm filter and thetemperature of the system was maintained at 25° C. The active agent(Healthin 1) extracted from the wheat grass had a retention time of 15.8min (see arrow on FIG. 1). This result was repeated in 5 extractions.Several blank runs were performed between each of the 5 sample runs toprevent residual chromatography corresponding to the elution of theactive component.

Active component detection was performed with an amperometric detectorLC-4C (BAS). The microbore column was coupled directly to the detectorcell to minimize the dead volume. The electrochemical detection systemused a glassy carbon-working electrode (3 mm) and a 0.02 Hz filter (500mV; range 10 nA). The cell volume was reduced by a 16 μm gasket. Thechromatographic system was controlled by the Waters Millenniumchromatography Manager V3.2 software and the chromatograms wereintegrated with Chromatograph software (Waters). The concentration wasextrapolated from the peak area. The average concentration in the 5samples was 1 μg/gm dry weight. Blank runs between determinations failedto elicit carry over residue. The fractions from each of the 5 runs werecollected, dried and applied in the NO tissue assays described below.Results are illustrated in FIG. 1.

An alternative method of purification was performed by methanolextraction followed by HPLC purification on a Spherisorb column asfollows. One gram of wheat grass, Agropyron spp, was homogenized in 50%methanol, 50% purified water, extracted with 50% methanol, and dried byspeed vacuum. The sample was stored at −20° C. HPLC purification wascarried out with a two solvent system: Buffer A was composed of 10 mM1-heptane sulfonic acid, sodium salt and 10 mM sodium phosphatemonobasic water, pH 3; Buffer B was composed of 10 mM 1-heptane sulfonicacid, sodium salt and 10 mM sodium phosphate monobasic, 50% methanol.The injection volume was 10 microliters. The running conditions were:0-10 min, 50% Buffer B; 10-20 min, Buffer B increased from 50 to 100%;25 min, 100% Buffer B; 35 min, 50% Buffer B. Fractions were collectedfrom 0 to 30 minutes after sample injection. The collected fractionswere dried by speed vacuum and maintained at −20° C. The active agentextracted from the wheat grass had a retention time of 16 min (see arrowon FIG. 1 a).

Example 2 Extraction of Healthin II from White Willow Bark

The identical procedure was performed with 0.02 grams (dry weight) ofwhite willow bark, Salix alba, The active agent (Healthin 2) extractedfrom the white willow bark had a retention time of 16.50 min. Theaverage concentration in the 5 samples sun was 0.3 μg/gm dry weight. SeeFIG. 2.

Example 3 Mass Spectrometric Identification of Active Agents fromAgropyron

The HPLC fraction, 1/100 microliters, containing the NO releasingactivity from the first purification detailed in Example 1 above wassubjected to nano electrospray ionization double quadrupole orthogonalacceleration Time of Flight mass spectrometry (Q-TOF-MS) on a MicromassQ-TOF system (Micromass, UK) as follows. One μl ofacetonitril/water/formic acid (50:49:1, v/v/v) containing the sample wasloaded in a gold-coated capillary Micromass F-type needle. The samplewas sprayed at a flow rate of 30 nl/min, giving an extended analysistime during which MS spectrum and several MS/MS spectra were acquired.During MS/MS, or tandem mass spectrometry, fragmentations are generatedfrom a selected precursor ion by collision-induced dissociation (CID).Since not all ions fragment with the same efficiency, the collisionenergy is typically varied between 20 and 35 V, so that the parent ionis fragmented into a satisfying number of different daughter ions.Needle voltage was set at 950 and cone voltage was set at 25. Theinstrument was operated in the positive mode. The results areillustrated in FIG. 3. Healthin I, the active agent isolated andpurified from the wheat grass sample, yielded major signals at 353.28and 119.05 daltons.

Example 4 Mass Spectrometric Identification of Active Agents from Salixalba

The identical procedure from Example 3 was performed with one gram ofwhite willow bark, Salix alba. The results are shown in FIG. 4. HealthinII, the active agent isolated and purified from white willow barksample, yielded major signals at 353.28, 192.15, 109.09 and 97.1daltons.

Example 5 Agropyron Extract Stimulation of NO in Pedal Ganglia andEndothelial Cells

Ten Mytilus edulis pedal ganglia, dissected from live animals, wereplaced in 1.5 ml Eppendorf tubes with 990 μl of phosphate buffer saline(PBS). Cultured human vein endothelial cells (ATCC # CRL 1730) werewashed in PBS at 4° C. The vein endothelial cells were grouped intopatches of approximately 10⁶ cells each and placed in 990 μl of PBS at4° C. One gram of dried wheat grass, Agropyron spp, was purified by HPLCas detailed above and the fraction corresponding to the retention timeof the Healthin 1 was collected and dried. The fraction was thenreconstituted in 20 μl PBS. 10 μl were added to the tubes containing theganglia or the endothelial cells or PBS alone (control). NO productionwas determined using a Mark II isolated nitric oxide meter (WorldPrecision Instruments, Sarasota, Fla.) fitted with a 200 μM sensor. If aresponse was detected in the tube containing PBS alone, the amount wassubtracted from the amounts detected in the tubes containing the tissuesamples.

The results are shown in FIGS. 5 and 6. The pedal ganglia tube cellsreleased 17 nM NO (FIG. 5), the human endothelial cells released 91 nMNO (FIG. 6). The identical volume added to the control tube resulted inthe production of <3 nM NO.

Example 6 Salix alba Extract Stimulation of NO in Pedal Ganglia andEndothelial Cells

The procedure detailed in Example 5 above was performed with onemilligram of the agent purified from the white willow bark, Salix alba,from Example 2. The results are shown in FIGS. 7 and 8. The pedalganglia tube cells released 19 nM NO (FIG. 7), the human endothelialcells released 87 nM NO (FIG. 8). The identical volume added to thecontrol tube resulted in the production of <3 nM NO.

Example 7 Analysis of Plants of Various Species for NO Release

Employing the isolation and purification techniques described above, avariety of herbaceous plants were analyzed for their ability to releasecNOS- derived nitric oxide in the pedal ganglia and in publiclyavailable SK-N-MC (ATCC # HBT-10) and PC-12 (ATCC # CRL 1721) cells.These results are set forth in Tables I, II, and III below. In Table I,a plus sign indicates detection of at least 1 nM nitric oxide. A minussign indicates no detection or detection of less than 1 nM nitric oxide.In Table II, results in the SK-N-MC cell line are set forth; theconcentration of plant material used and the quantity of NO detected isindicated. In Table III, results are set forth for the identicalprocedures performed using the ganglia cell line. The types of plantmaterials employed are indicated, for example flowers, leaves, roots,rhizomes, stems, bark. Where not specified, leaves were employed. FIG. 9shows an exemplary result.

TABLE I NO determination of ganglia, SK-N-MC and PC-12 cells treatedwith various plant extractions. Blank indicates plant not tested in thatcell line. Ganglia SK-N-MC PC-12 Allium vineale (Garlic) — + Salix alba(White willow) bark + + Agropyron (Wheat grass) + + Petroseliniumcrispum or Carum — + petroselinum (Parsley) Taraxacum officinale(Dandelion) + — Sesamum indicum (Sesame, Gin sum) + leaves Medicago spp.(Alfalfa) + Piper methysticum (Kava) + Anthemis spp. (Chamomile) +++ +Turnera diffusa (Damian) + Verbascum densiflorum (Mullein) + Marantaarundinaceae (Arrowroot) roots — Lavandula angustifolia (Lavender)flower — Ocimum spp. (Sweet basil) — Artemesia dracunculus (Tarragon)leaves — Aloe vulgaris or A. barbadensis (Aloe) — — leaves Vacciuiummembranaceum (Bilberry) — — Brassica spp. (Cabbage) — — Daucus carota(Carrot) — — Zea mays flowers (corn silk) — — Echinacea (Coneflower) — —Lactuca spp. (Lettuce) — — Tabebuia impetiginosa, T. avellanedai, — —Tecoma curialis (Pau d'arco) Mentha piperita (Peppermint) — — Rubus spp.(Raspberry) — — Rosmarinus officinalis (Rosemary) — — Salvia spp. (Sage)— — Equisetum hyemale (Shave grass) — — Ulmus rubra, Fremontodendron — —californicum (Slippery elm) bark Phaseolus spp. (String bean) — — Thymusspp. (Thyme) — —

TABLE II NO determination of SK-N-MC cells treated with various plantextractions Concentration Results (nM) Ocimum spp. (Basil)   6 mg ofcrude extraction 31 Verbascum densiflorum   6 mg of crude extraction Noeffect (Mullein) Tumera diffusa (Damian)   6 mg of crude extraction Noeffect Maranta arundinaceae   6 mg of crude extraction 31 (Arrowroot)root Coriandrum sativum   6 mg of crude extraction 172  (Cilantro)Artemesia dracunculus   6 mg of crude extraction 135  (Tarragon)Lavendula augustifolia   6 mg of crude extraction 48 (Lavender) flowerMentha pulegium   6 mg of crude extraction 66 (Pennyroyal) Quercetine*  6 mg of crude extraction 14 Piper methysticum 1.5 mg 108  (Kava)Anthemis spp. 1.5 mg 31 (Chamomile) Centella asiatica 1.5 mg Reactive inPBS (Gotu kola) Scutellaria lateriflora 1.5 mg Negative (Skullcap) Ginkobiloba (Ginko) 1.5 mg Reactive in PBS Hypericum perforatum 1.5 mgNegative (St John's Wort) Urtica dioeca 1.5 mg Negative (Common nettle)*Quercetine (from Sigma Chemicals) is a plant flavanoid found in manyplants, and especially in fruits.

TABLE III NO determination of ganglia cells treated with various plantextractions Anthemis spp. (Chamomile) 6 mg of crude extraction 67 nMPiper methysticum (Kava) root 6 mg of crude extraction 13 nM Turneradiffusa (Damian) 6 mg of crude extraction 22 nM Verbascum densiflorum(Mullein) 6 mg of crude extraction 15 nM Ocimum spp. (Basil) 6 mg ofcrude extraction 19 nM

Example 8 Grape Skin Extraction and NO Release

Ten grams (wet weight) of black grape skins, Vitis vinifera, were placedin a 50 ml Falcon tube with 15 ml of a 1:1 mixture of methanol orethanol and water. The tubes were shaken overnight at room temperatureand the resulting extracts were aliquoted, 1 ml per tube, into twelve1.5 ml Eppendorf tubes. The tubes were evaporated to dryness in aspeedvac and then reconstituted in 1 ml phosphate buffered saline (PBS)solution. 10 μg o this solution was used to treat the invertebratenervous tissue pedal ganglia (see Example 5, above) and NO release wasmeasured in real time by an amperometric probe specific for themeasurement of NO. Grape skin extracted in methanol caused a release ofNO within 15 seconds of treatment (see FIG. 10) whereas grape skinextracted in ethanol did not (within the same time period). NO releasewas not observed when the extract (either methanol or ethanol extracted)was added to PBS alone.

Example 9 Anti-Microbial Effects of Extracts on Cells

A dried, powdered, formulation of a 1:1 mixture of the wheat grassextract and white willow bark extract prepared in Example 1 above wastested for its ability to inhibit bacterial growth in culture. Theformulation was reconstituted in 10 ml of LB broth (AmershamBiosciences, Inc.). The broth was then inoculated with E. coli bacteriaand incubated for 5 and 24 hours at 37° C. 20 μl of the cultures werestreaked on LB-agar plates and incubated overnight at 37° C. There wasno growth observed in the 5 and 24 hours bacterial cultures as comparedto the control (LB broth alone).

An additional control experiment was conducted with the knownantibacterial agent, SNAP. One μg/ml SNAP was added to LB broth. Thebroth was then inoculated with E. coli bacteria and incubated for 5 and24 hours at 37° C. 20 μl of the cultures were streaked on LB-agar platesand incubated overnight at 37° C. Bacterial growth was decreased in theSNAP culture at 5 and 24 hours, as compared to the control.

This experiment demonstrates that the wheat grass/white willow extractof the invention exhibits greater antibacterial activity than the knownantibacterial agent SNAP.

Example 10 Anti-Cancer Effects of Extracts on SK-N-MC Cells

SK-N-MC cells were incubated with either garlic (Allium vineale) orparsley (Petroselinium crispum) extractions, 0.005 g/ml in RPMI media,for two days. The cells were then stained with Trypan Blue indicator(Invitrogen Corp.) and observed under a research microscope at 200×.Healthy cells do not allow this indicator to enter the cell wall whereascells which turn blue are dead or dying because the reagent has enteredthe cytoplasm. Microscope observation of both garlic and parsley treatedcells indicated almost 100% of the cells were dead. Similar results wereobserved with 1 N solutions of Mullein (Verbascum densiflorum), Kava(Piper methysticum), Chamomile (Anthemis spp.), and Damian (Turneradiffusa). Other plant extracts prepared and tested in a similar mannerthat induced cell death in SK-N-MC cells were Bilberry (Vacciniummyrtillus), Enchinaceae purpurae, Garlic (Allium vineale), Goldenseal(Hydrastis candensis), Parsley (Petroselenium crispum or C.petroselenium), Paul d'arco bark (Tabebuia impetiginosa), Rosemary(Rosmarinus officinalis), Slippery elm (Ulmus rubra or Fremontodendroncalifomicum), and White willow bark (Salix alba). The strongestanti-cancer effects were seen with garlic and parsley.

Plant extracts prepared and tested in the same manner that exhibited noanti-cancer effect on SK-N-MC cells included Raspberry (Rubus spp.),Peppermint (Mentha piperita), Shave grass (Equisetum hyemale), cornsilk(Zea mays flowers), Dandelion (Taraxacum officinale), Alfalfa (Medicagospp.), Thyme (Thymus spp.) and Slippery Elm (Ulmus rubra andFremontodendron califomicum).

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1. A pharmaceutical composition for stimulating nitric oxide productionin mammalian cells, said pharmaceutical composition comprising aneffective amount of extract of Salix alba, wherein the Salix albaextract is prepared by a process comprising: (a) homogenizing driedSalix alba in an acidic solution; (b) preparing a supernatant byseparating solid material from the homogenized acidic solution usingalcohol extraction and centrifugal filtration; (c) drying thesupernatant; (d) preparing an elute by dissolving the supernatant in anaqueous solution containing trifluroacetic acid and extracting a solidphase; and (e) purifying the elute using high performance liquidchromatography to obtain said extract.
 2. The pharmaceutical compositionaccording to claim 1, the process further comprising: identifying andcharacterizing the extract using mass spectrometric analysis.
 3. Thepharmaceutical composition according to claim 1, wherein the extractstimulates nitric oxide production in mammalian cells.
 4. Thepharmaceutical composition according to claim 1, wherein the extractcontains a first compound with a molecular weight of 263.3 Daltons and asecond compound with a molecular weight of 356.5 Daltons and a thirdcompound with a molecular weight of 337.5 Daltons and a fourth compoundwith a molecular weight of 354.4 Daltons.
 5. The pharmaceuticalcomposition according to claim 1, wherein the composition has at leastone component selected from a group consisting of: a component have amolecular weight of 353.28 Daltons, a component have a molecular weightof 109.09 Daltons, and a component have a molecular weight of 97.1Daltons.
 6. The pharmaceutical composition according to claim 1, whereinthe composition has the ability to stimulate nitric oxide release in therange of 15 nM to 100 nM in pedal ganglia cells.
 7. The pharmaceuticalcomposition according to claim 1, wherein the composition has theability to stimulate nitric oxide release in the range of 50 nM to 100nM in endothelial cells.
 8. The pharmaceutical composition according toclaim 1, wherein said extract displays a single major peak on highperformance liquid chromatographic analysis in 10 nM sodium chloride,0.5 mM EDTA, 100 mM sodium acetate and 50% acetonitrile, pH 5.0.
 9. Amethod of preparing a Salix alba extract, comprising: homogenizing driedSalix alba in an acidic solution; preparing a supernatant by separatingsolid material from the homogenized acidic solution using alcoholextraction and centrifugal filtration; drying the supernatant; preparingan elute by dissolving the supernatant in an aqueous solution containingtrifluroacetic acid and extracting a solid phase; and purifying theelute using high performance liquid chromatography to obtain saidextract.
 10. The method according to claim 9, further comprising:identifying and characterizing the extract using mass spectrometricanalysis.
 11. The method according to claim 9, wherein the extractstimulates nitric oxide production in mammalian cells.
 12. The methodaccording to claim 9, wherein the extract contains a first compound witha molecular weight of 263.3 Daltons and a second compound with amolecular weight of 356.5 Daltons and a third compound with a molecularweight of 337.5 Daltons and a fourth compound with a molecular weight of354.4 Daltons.
 13. The method according to claim 9, wherein the extractincludes at least one component selected from a group consisting of: acomponent have a molecular weight of 353.28 Daltons, a component have amolecular weight of 109.09 Daltons, and a component have a molecularweight of 97.1 Daltons.